This invention relates to carbohydrate binding proteins. More specifically, the invention relates to a group of proteins referred to as lectins, which are associated with tumor cells and which have a binding affinity for carbohydrates such as galactose. Most specifically, the invention relates to a particular amino acid sequence in the protein which is responsible for its galactose binding activity. In particular embodiments, the present invention includes assays for the presence of tumor cells as well as therapies for inhibiting metastasis of tumor cells.
A major thrust in metastasis research has been the search for cellular genes and other epigenetic factors which control the metastatic cascade. It has been determined that there is a close correlation between tumor cell surface receptors and metastasis of those cells. This research has let to the supposition that cellular interactions are influenced by cell surface components; however, a detailed structural analysis of such cellular components has not heretofore been undertaken.
In accord with the present invention, it has been found that particular tumor cells include a class of proteins termed lectins on their surface, and those lectins bind to galactose. Accordingly, within the context of this disclosure, such lectins will be collectively referred to as carbohydrate binding proteins (CBP). Tumor progression can be delineated as either suppressed or enhanced expression of a relatively limited number of cell proteins, and the CBPs have been found to increase in number as a tumor progresses to metastasis. Consequently, the CBPs play a pivotal role in malignant biochemical transformation. It is believed that CBP may mediate the interaction between adjacent cells and cell matrix recognition by binding complementary glyco-conjugates.
The amino acid sequence of a number of CBPs has previously been determined; however, the precise structure of the active portion of CBPs responsible for the galactose binding was heretofore unknown. In general, it has been found that the carbohydrate binding protein isolated from different tissues by affinity chromatography appears to constitute two different classes of peptides. One class of peptides has a molecular weight of about 14,000 dalton. The other class has a molecular weight ranging between 20,000-35,000 daltons. It has also been found that CBPs obtained from different species of animals often show immunological cross activity, suggesting structural similarities. Galactose binding proteins of approximately 14,000 and 34,000 daltons have been extracted and cloned from different tissues, and from various species. These materials have been sequenced and the homology range is from 40-80%. Therefore, it will be appreciated that data developed in animal models, such as the mouse or rat, is highly applicable to another species including humans.
It has been found that a number of different tumor cells contain CBPs that are very similar to those isolated from normal cells having sugar binding specificity. Further studies have shown that neoplastic transformation is associated with the concomitant expression of an additional, unique CBP species having a molecular weight of approximately 34 kilodalton designated as L-34; see, Lotan, R and Raz A. Cancer research 43:2088 (1983).
Other families of carbohydrate-binding proteins that share common binding specificity for sugars such as galactose exist, despite the fact that such proteins are very diverse in structure and function. Included are a group of 14 kilodalton galactoside binding lectins, a 64 kilodalton component of the elastin receptor, the 55 kilodalton ectosialyltransfarese of Hodgkins disease, the 43 kilodalton human actin-binding brain lectin, the 50 kilodalton rat testis galactosyl receptor, the murine and human tumor associated 34 kilodalton lectin, the 35 kilodalton fibroblast carbohydrate-binding protein, the IgE-binding protein, the 32 kilodalton macrophage non-integrin laminin-binding lectin and the rat, mouse and human 29 kilodalton galactoside-binding lectin. All of these diverse polypeptides have been found to share significant homology and are designated carbohydrate-binding proteins within the context of this disclosure.
Based upon studies of the various tumor cells it has been found that CBPs play a role in cellular interactions in vivo. These reactions are important for the formation of emboli and the arrest of circulating tumor cells leading to the development of metastatic lesions.
In accord with the present invention, the active site on the carbohydrate-binding protein responsible for galactose affinity has been identified. Furthermore, it has been found that this particular amino acid sequence is highly homologous throughout a number of species. For example, the site approaches 90% homology in mouse and human tissues. For this reason, results obtained from studies in mice are highly predictive of human results. In accord with a further aspect of the present invention, there is provided a highly sensitive blood test for the presence of potentially metastatic tumor cells, which is based upon detecting the presence of the particular galactose-binding site. The present invention also makes possible, and includes therapeutic methods for inhibiting metastases, based upon the properties of the galactose receptor. These and other advantages of the present invention will be apparent from the drawings, discussion and description which follow.
In accord with the conventions codified in 37 C.F.R. 1.821, the abbreviations used for amino acids in the following disclosure and claims shall be:
Alaxe2x80x94alanine
Argxe2x80x94arginine
Asnxe2x80x94asparagine
Aspxe2x80x94aspartic acid
Cysxe2x80x94cysteine
Gluxe2x80x94glutamic acid
Glnxe2x80x94glutamine
Glyxe2x80x94glycine
Hisxe2x80x94histidine
Ilexe2x80x94isoleucine
Leuxe2x80x94leucine
Lysxe2x80x94lysine
Metxe2x80x94methionine
Phexe2x80x94phenylalanine
Proxe2x80x94proline
Serxe2x80x94serine
Thrxe2x80x94threonine
Trpxe2x80x94tryptophan
Tyrxe2x80x94tyrosine
Valxe2x80x94valine
There is disclosed herein a galactose-specific, carbohydrate binding protein. The protein includes the amino acid sequence consisting essentially of:
Ile, Val, Cys, Asn, Thr, Lys, Leu, His, Asn, Asn, Trp, Gly, Arg, Glu, Glu, Arg, Gln, Ser, Val, Phe, Pro, Phe, Glu, Ser, Gly.
In a still further embodiment, the protein includes the longer amino acid sequence:
His, Phe, Asn, Pro, Arg, Phe, Asn, Glu, Asn, Asn, Arg, Arg, Val, Ile, Val, Cys, Asn, Thr, Lys, Leu, His, Asn, Asn, Trp, Gly, Arg, Glu, Glu Arg, Gln, Ser, Val, Phe, Pro, Phe, Glu, Ser, Gly.
In another embodiment, the method includes an immunotherapeutic method for generating antibodies in animals to cells which include a galactose specific carbohydrate binding protein. The method includes the steps of providing a polypeptide which includes the amino acid sequence:
Ile, Val, Cys, Asn, Thr, Lys, Leu, His, Asn, Asn, Trp, Gly, Arg, Glu, Glu, Arg, Gln, Ser, Val, Phe, Pro, Phe, Glu, Ser, Gly;
injecting the polypeptide into an animal so that an immune response occurs wherein the animal generates antibodies to the peptide. In some embodiments, adjuvants may be employed to increase antibody production. In other embodiments, antibodies may be raised in one animal and subsequently transferred to another for therapy.
In accord with another embodiment of the present invention, there is provided an assay method for determining the presence of metastic cells in an animal""s bloodstream. The method includes the steps of providing a support member having a binding affinity for a carbohydrate binding protein which includes the amino acid sequence:
Ile, Val, Cys, Asn, Thr, Lys, Leu, His, Asn, Asn, Trp, Gly, Arg, Glu, Glu, Arg, Gln, Ser, Val, Phe, Pro, Phe, Glu, Ser, Gly;
contacting the support member with a fluid sample from the animal, maintaining the fluid sample in contact with the support member so that any of said carbohydrate binding protein present in the fluid sample will bind to the support; and detecting the bound protein, whereby the presence of the protein is indicative of the presence of metastatic cells in the animal. In one particular embodiment, the support member includes pectin adhered thereto. The step of detecting the bound protein may comprise contacting the bound protein with an antibody having affinity for the protein and subsequently detecting that protein. Also included is an assay kit for carrying out the analysis.
In another embodiment, the present invention includes a therapeutic method for inhibiting metastasis of a tumor cell of the type expressing a galactose binding protein and the surface thereof. The method comprises contacting the cell with the therapeutic agent which comprises galactose bound to a polymer. The polymer is preferably of a molecular weight in excess of 10 kilodaltons. The galactose may be part of a polysaccharide chain bound to the polymer.